Protein Protocols Protein Protocols

Immunogold 401
Table 2
Effect of Temperature on Enhancement Time and Enhancement
Reagent Stability
Temperature Typical enhancement Typical stability
(°C) time (min) time (min)
16 27–45 >80
18 22–38 >70
20 20–35 >55
22 18–33 >45
24 16–27 >40
5. If desired, the incubations in immunogold reagent can be extended to overnight when
concentrations of 1:100 to 1:400 are used.
6. If, after performing the experiment, there is a complete absence of signal, the reactivity of
the immunogold reagent with the primary antibody should be checked by performing a
dot-blot assay. Prepare a dilution series of primary antibody, for example, from 250 to
0.5. ng/µL. Spot out 1 µL onto the membrane, and allow to air-dry. Proceed with the
appropriate immunogold silver staining using the blocking, washing and incubation conditions
as described in Subheadings 3.2. and 3.3. (see Table 1).
4.3. Silver Enhancement
7. The enhancement reagents are extremely sensitive to the purity of the water used. Low
quality water results in the formation of precipitates that reduce the reactivity of the
enhancement and can lead to high backgrounds. In addition, glassware contaminated
with heavy metals in elemental form or as heavy metal salts will decrease the performance
of the enhancer reagents.
8. The silver enhancement reagents are prepared by mixing the enhancer and initiator solutions
in equal quantities. The mixture is usable only over a defined time period (see
Table 2), so it is important that the components be combined immediately before use.
There is no need to shield the silver enhancer from normal daylight.
9. Both the enhancement time and the stability of the silver-enhancement mixture vary considerably
with ambient temperature. A typical enhancement time for most blotting experiments,
of 15–40 min at room temperature (22°C) is recommended. For some applications,
it may be necessary to extend the enhancement time, and for others, to shorten it. At room
temperature, there is a comfortable safety margin to enable maximum enhancement before
there is any danger of self-nucleation of the enhancer reagent occurring.
10. When a very strong amplification signal is desired, it is possible to perform a second
silver-enhancement step before self-nucleation starts. In this case, the blot is subjected to
silver enhancement for 30 min at room temperature (22°C). It is then rinsed with distilled
water and immersed in a freshly prepared silver-enhancement solution for another
20–30 min. The increase in signal slows down considerably with time. After enhancement,
the blot should be washed in distilled water and dried.
4.4. General Protein Staining with AuroDye Forte
11. Owing to the high sensitivity of AuroDye forte, special care needs to be taken to avoid
background staining. It is important to wear gloves when handling gels and blots. Where
possible, handle blots by their edges with forceps, since gloves can leave smears. High
quality chemicals should be used throughout.